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| Status |
Public on May 06, 2023 |
| Title |
MC_EryPre_Rep2 |
| Sample type |
SRA |
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| Source name |
Cord Blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Cord Blood
cell line: Erythroid precursor
cell type: Erythroid precursor
genotype: pooled
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
About 100,000 FACS-purified cells were used for each reaction. In brief, after fixation with 3mM DSG for 30 min followed by 1% formaldehyde for another 10 min, crosslinked cells were permeabilized in ice-cold Micro-C Buffer #1 (50mM NaCl, 10mM Tris-HCl pH = 7.5, 5mM MgCl2, 1mM CaCl2, 0.2% NP-40, 1x Protease Inhibitor Cocktail) for 20 minutes. Chromatin from permeabilized cells was digested by 50U micrococcal nuclease at 37°C for 10 minutes. After digestion, chromatin fragments were then subjected to dephosphorylation, phosphorylation, end-chewing process by T4 PNK and Klenow Fragment in end-repair buffers (50mM NaCl, 10mM Tris-HCl pH = 7.5, 10mM MgCl2, 100ug/mL BSA, 2mM ATP, 5mM DTT, no dNTPs) at 37°C for 30 minutes. Blunt-end and biotin incorporation was achieved by adding biotin-dATP, biotin-dCTP, dGTP, and dTTP and incubated at 25°C for 45 minutes. Chromatin was ligated by T4 DNA ligase at room temperature for 2.5 hours. Unligated ends containing biotin-dNTPs were then removed by exonuclease III in 37°C for 15 minutes.
After reverse-crosslinking, DNA was purified and gel size-selected for dinucleosomal DNA at 200 to 400 bp. DNA fragments containing biotin were immobilized on MyOne Streptavidin C1 beads (ThermoFisher Cat. #: 65602), and then library preparation was performed using the DNA library preparation kit (Vazyme, Cat.# ND607) as per the manufacturer’s protocol. Two biological replicates per condition were sequenced in a NovaSeq 6000 (S4, PE150, Illumina) by Novogene (Beijing).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The raw reads of Micro-C library were mapped with hg19 and further filtered with HiC-Pro (version 3.10.0, https://github.com/nservant/HiC-Pro). Pairs with multiple hits, MAPQ <10, singleton, dangling end, self-circle, and PCR duplicates were removed. Paired reads with distances shorter than 100 bp were also disregarded.
Correlation of replicates was calculated by Stratum-adjusted Correlation Coefficient (SCC) method with HiCRep (http://github.com/qunhualilab/hicrep) at 10 kb, 20 kb, 50 kb and 100 kb. All biological replicates were processed individually to assess quality and then combined for further analyses and visualization. The valid Micro-C contacts matrices were then normalized by using K-R method and corresponding .cool files were generated at 200 bp, 400 bp, 600 bp, 800 bp, 1 kb, 2 kb, 4 kb, 10 kb, 20 kb, 100 kb, 200 kb bin size. Visualization of .cool files were conducted on Higlass 3D genome server (http://higlass.io).
We utilize Mustache (https://github.com/ay-lab/mustache to call loops with balanced contact matrices at resolutions of 400 bp, 600 bp, 800, 1 kb, 2 kb, 4 kb, 10 kb, and 20 kb using the calling options --pThreshold 0.1 --sparsityThreshold 0.88 -–octaves 2. We then combined all loops at different resolutions. If a loop was detected at different resolutions, we retained the precise coordinates in finer resolutions. Coolpup (https://github.com/open2c/coolpuppy) has implemented the APA function for the .cool file. To further filter and classify loops, we first defined +-2.5 kb of gene TSS as promoter (P) and Non-TSS H3K27ac peaks in each stage (HSPC, Ery-Pro and Ery-Pre) as enhancers (E). GenomicInteractions (https://github.com/ComputationalRegulatoryGenomicsICL /GenomicInteractions) was used to designated P-P, E-P and E-E loops in respective stages.
Assembly: hg19
Supplementary files format and content: mcool
Supplementary files format and content: bedpe: loop files
Library strategy: Micro-C
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| Submission date |
Oct 04, 2022 |
| Last update date |
May 06, 2023 |
| Contact name |
Dong Li |
| E-mail(s) |
lidongpaul@gmail.com
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| Organization name |
Peking University
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| Department |
College of Life Science
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| Lab |
Hsiang-Ying (Sherry) Lee
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| Street address |
No.5 Yiheyuan Road, Haidian District,
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE214808 |
Stage-specific erythroid cell three-dimensional chromatin architecture and transcription factors binding provide insight of human erythropoiesis [MicroC] |
| GSE214811 |
Stage-specific erythroid cell three-dimensional chromatin architecture and transcription factors binding provide insight of human erythropoiesis |
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| Relations |
| BioSample |
SAMN31015780 |
| SRA |
SRX17706200 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6616197_MC_EryPre_Rep2.mcool |
5.3 Gb |
(ftp)(http) |
MCOOL |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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